266 ANN IST SUPER SANITÀ 2010 | VOL. 46, NO. 3: 266-273 DOI: 10.4415/ANN_10_03_07 ERN Quantitative real-time PCR of enteric C ON viruses in influent and effluent samples C H LT from wastewater treatment plants in Italy A E H Giuseppina La Rosa, Manoochehr Pourshaban, Marcello Iaconelli and Michele Muscillo OF Dipartimento di Ambiente e Connessa Prevenzione Primaria, Istituto Superiore di Sanità, Rome, Italy SSUES I L Summary. The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in elimi- A nating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction) analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologi- RONMENT VI cally important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses N (GI and GII) and compared to classical bacterial indicators of fecal contamination. The concentration E of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adeno- virus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water, pointing to potential public health risks through the release of these viruses into the environment. A better understanding of viral presence and resistance to sewage purification processes have the potential of contributing to the effective management of risks linked to the recycling of treated wastewater, and its discharge into the environment. Key words: norovirus, adenovirus, enterovirus, sewage, real-time quantitative PCR assays. Riassunto (Determinazione quantitativa mediante real-time PCR di virus enterici in campioni di acque reflue affluenti ed effluenti da impianti di trattamento dei liquami in Italia). La prevalenza dei virus enterici nei liquami urbani, la loro rimozione durante i trattamenti di depurazione delle acque reflue, e rischi po- tenziali per la salute legati alla contaminazione degli ambienti idrici o al riutilizzo delle acque depurate, sono argomenti di grande interesse in microbiologia ambientale. In questo studio abbiamo stimato la concentrazione di virus enterici, in campioni di reflui grezzi e depurati di 5 impianti di trattamento di reflui urbani localizzati in Roma e dintorni, utilizzando metodologie quantitative molecolari (TaqMan real-time PCR). Sono stati analizzati tre gruppi di virus enterici responsabili di epidemie di origine idrica (adenovirus, enterovirus e norovirus GI/GII), parallelamente ai classici indicatori batterici di contami- nazione fecale. Le concentrazioni virali più elevate sono state rilevate per gli adenovirus, sia nei reflui non depurati sia in quelli trattati. Nei reflui grezzi sono state osservate le seguenti concentrazioni medie: adenovirus > norovirus GI > norovirus GII > enterovirus; negli effluenti depurati: adenovirus > noro- virus GI > enterovirus > norovirus GII. L’efficienza di rimozione virale variava dal 35% (enterovirus) al 78% (norovirus GI), mentre per gli indicatori batterici raggiungeva il 99%. Poiché i metodi molecolari non forniscono indicazioni circa l’infettività dei virus rilevati, si è proceduto a valutare la capacità di replicazione delle particelle virali (limitatamente agli enterovirus) nei liquami depurati utilizzando un sistema integrato colture cellulari/real-time PCR. L’analisi ha rilevato particelle virali infettive, indicando potenziali rischi per la salute pubblica legati alla contaminazione dei corpi idrici recettori. Una migliore comprensione della resistenza dei virus ai processi di depurazione è importante per la gestione dei rischi legati al riutilizzo delle acque reflue trattate, o alla loro immissione nell’ambiente. Parole chiave: norovirus, adenovirus, enterovirus, liquami, PCR quantitativa real-time. INTRODUCTION Most treated wastewater, as well as untreated sewage, Enteric viruses are largely excreted through fecal flow into the water environment and have the poten- matter, and current methods of sewage treatment tial to impact agricultural, recreational and drinking do not always effectively remove these organisms [1]. waters. Currently, bacterial indicators such as fecal Address for correspondence: Giuseppina La Rosa, Dipartimento di Ambiente e Connessa Prevenzione Primaria, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: [email protected]. QUANTIFICATION OF ENTERIC VIRUSES AT WTPS 267 coliforms and enterococci are commonly used to method combining the sensitive conventional quan- assess water quality. These bacteria are simple and titative PCR with an infectivity assay. ERN inexpensive to monitor, but have been shown to be C less than ideal as indicators of fecal pollution. In ON METHODS C fact, enteric human-pathogenic viruses are generally H more resistant than enteric bacteria to current meth- Sample collection LT A ods of wastewater treatment [2, 3]. Different studies Five different municipal WTPs were chosen, all E having found that, despite treatment, enteric viruses situated in central Italy, within 30 km of each oth- H OF persist in high levels in wastewaters [4-7], proposed er. The WTPs in question are conventional activat- the use of a viral indicator of wastewater contami- ed sludge plants which receive waters from urban nation. Specifically, enteroviruses (EVs) and adeno- areas. Wastewater treatment typically includes grid SSUES viruses (AdVs) have been suggested as indicators for separation, primary sedimentation, secondary bio- I L monitoring the human fecal contamination of wa- logical treatment and final disinfection with chlo- A ter and for determining the efficacy of disinfection rine, before effluents are discharged into the water treatments [8]. The monitoring of wastewater treat- environment. Daily flows in these plants range from ment plants (WTP) may prove a suitable approach 4800 to 750 000 cubic meters, and design capacity RONMENT for the study of circulating viruses in their respective from 60 000 to 1 100 000 population equivalents. VI service areas [9], and the persistence of such viruses No industrial wastewater is treated in any of these N in treated effluents. facilities. E The objective of the present study was to assess, A total of 50 grab samples were collected (25 in- in both raw and treated sewages, the concentration flows and 25 outflows, 50 mL each). Sampling was of three epidemiologically important, waterborne carried out monthly, from May to September 2007. enteric viruses, AdVs, EVs and noroviruses (NoV Samples were collected in the morning on sunny GI and NoV GII), to gain insight into the infectious days, stored in sterile 50 mL Falcon tubes kept in potential of viral contamination of treated sewage, thermal bags at 4 °C, and delivered to the labora- and to obtain a rough estimate the efficacy of dis- tory, where they were processed within 24 hours. infection treatments. For the sake of reference, we also tested and quantified two enteric bacteria com- Concentration of viruses and RNA extraction monly used as fecal indicators, Escherichia coli and Viruses were concentrated as previously described enterococci. [16]. Briefly, samples were centrifuged at 3000 ×g at 4 EVs are single-stranded RNA, non-enveloped vi- °C for 10’. Supernatants were centrifuged at 200 000 ruses. Symptoms of EV infection include mild up- × g for 2 h. The pellets were resuspended in 1 mL of per respiratory illness, febrile rash, aseptic meningi- phosphate-buffered saline and the suspension aliq- tis, pleurodynia, encephalitis, acute flaccid paralysis, uoted and stored at -80 °C until genome extraction. paralytic poliomyelitis, gastroenteritis, myocarditis, To assess virus recovery, we spiked 50 mL of dis- pericarditis and diabetes [10, 11]. tilled water with 1.91 × 108 genome copies (GC) of Human AdVs are double-stranded DNA, non-en- Echovirus 2 (Cornelis strain, american type culture veloped viruses, with 52 serotypes grouped into six collection, ATCC, VR-32) and computed the ratio of species, from A to F. Symptoms of AdV infection GCs after/before concentration by real-time PCR. include gastroenteritis, pharyngitis, pneumonia, RNA and DNA were isolated from 100 µl of viral conjunctivitis, and meningoencephalitis [12]. pellet using the NucliSens miniMAG (BioMérieux NoVs, the most commonly identified cause of Italia, Florence, Italy) nucleic acid isolation kit, acute nonbacterial gastroenteritis in all age groups, based on the Boom nucleic acid extraction method contain a single-stranded, positive-sense RNA ge- [17] in combination with magnetic silica beads. A nome [13, 14]. These viruses are members of the NucliSens miniMag extractor (BioMérieux Italia, Caliciviridae family, and are classified into five ge- Italy) was used to collect and wash the magnetic nogroups, GI-GV [15]. silica particles, and to recover nucleic acids from the Each group